Preconcentration and Determination of Mefenamic Acid in Pharmaceutical and Biological Fluid Samples by Polymer-grafted Silica Gel Solid-phase Extraction Following High Performance Liquid Chromatography

Mefenamic acid is a nonsteroidal anti-inflammatory drug (NSAID) that has analgesic, anti-infammatory and antipyretic actions. It is used to relieve mild to moderate pains. Solid-phase extraction of mefenamic acid by a polymer grafted to silica gel is reported. Poly allyl glycidyl ether/iminodiacetic acid-co-N, N-dimethylacrylamide was synthesized and grafted to silica gel and was used as an adsorbent for extraction of trace mefenamic acid in pharmaceutical and biological samples. Different factors affecting the extraction method were investigated and optimum conditions were obtained. The optimum pH value for sorption of mefenamic acid was 4.0. The sorption capacity of grafted adsorbent was 7.0 mg/g. The best eluent solvent was found to be trifluoroacetic acid-acetic acid in methanol with a recovery of 99.6%. The equilibrium adsorption data of mefenamic acid by grafted silica gel was analyzed by Langmuir model. The conformation of obtained data to Langmuir isotherm model reveals the homogeneous binding sites of grafted silica gel surface. Kinetic study of the mefenamic acid sorption by grafted silica gel indicates the good accessibility of the active sites in the grafted polymer. The sorption rate of the investigated mefenamic acid on the grafted silica gel was less than 5 min. This novel synthesized adsorbent can be successfully applied for the extraction of trace mefenamic acid in human plasma, urine and pharmaceutical samples.


Introduction
Mefenamic acid is a non-steroidal antiinflammatory drug used to treat pain, including menstrual pain. It is typically prescribed for oral administration. It is usually available in 100 mg, 250 mg, 500 mg tablets or 250 mg capsules. Mefenamic acid can cause side effects such as headache, vomiting, diarrea, haematuria, hematemesis. These symptoms appear while taking this medication. Recently, non-steroidal anti-inflammatory drugs are broadly used for treatment of patients with rheumatoid arthritis (1, 2). These compounds non selectively inhibit the two isoforms of the cyclooxygenase (COX-1 and COX-2) and so prevent the metabolism of cellular arachidonic acid (AA) and the upregulation of prostaglandin formation, which in other respects lead to an increase of vascular silica with (3-mercaptopropyl) trimethoxy silane was performed. The interactions between the mefenamic acid and the active sites in the polymer are frequently hydrogen bonding and have reversible character for sorption and desorption phenomena. This novel polymer grafted silica gel in batch-wise mode, was used for the pretreatment of trace mefenamic acid in human urine and plasma prior to the determination of concentration of mefenamic acid by high performance liquid chromatography (HPLC).
The stock solution of mefenamic acid was prepared by dissolving appropriate amount in deionized water. To adjust the pH of the solutions, acetic acid-acetate buffer or phosphate buffer were used wherever suitable.
The non-steroidal anti-inflammatory drugs are divided into eight groups according to their structures. Amongst these groups anthranillic acid derivatives are of great importance. Mefenamic acid (see scheme 1 for molecular structure), 2-[2, 3-dimethylphenyl) amino] benzoic acid, is an anthranillic acid derivative with anti-inflammatory properties (5, 6).
To promote the sensitivity and selectivity of an analytical method, development and application of a practical sample preparation procedure prior to quantification is of great importance. Solid -phase extraction is a very common method for rapid sample preparation. The versatility of this technique allows us to use it for many purposes, such as preconcentration, purification, separation and trace enrichment. In the last few years, there are many publications about the use of solid -phase extraction in pharmaceutical products (23,24). Amongst the solid -phase extraction sorbents, silica gel plays an important role, since it does not swell or strain and has a good mechanical strength. The surface of silica gel particles is heterogeneous, with a variety of silanol groups which are excellent groups for modification reactions. Surface modification by grafting polymer chain to solid substrate is a useful method for creation of materials which possess specific surface and structural properties which can be used for preconcentration of trace analytes.
In this research, the performance of a novel synthetic support in adsorption of mefenamic acid is evaluated. To achieve this goal, free-radical graft co-polymerization of N, N-dimethylacrylamide and a designed monomer, Allyl glycidyl ether/ iminodiacetic acid (AGE/IDA) onto modified Scheme 1. Structure of mefenamic acid. out on a Shimadzu HPLC, 1200 series, equipped with UV/Vis detector. Separations were carried out on C18 column (150×460 mm), using 50 mM solution of monobasic ammonium phosphate, and adjusted with 3M ammonium hydroxide to a pH of 5.0 as the buffer solution at a flow-rate of 1 mL per minute. The effluent from the column was monitored at 280 nm. The pH measurements were made by a Metrohm model 744 pH meter (Zolingen, Switzerland). Infrared spectra were recorded on a Jasco Fourier Transform infrared spectrometer (FTIR-410, Jasco Inc.Easton, Maryland). The UV/Vis spectra were obtained by JascoV-530 UV/Vis spectrometer (Jasco Inc. Easton, Maryland).

Method procedure
The degree of mefenamic acid sorption at different pH values was determined by the batch equilibration technique. To study this effect, a set of 10 µg/mL solutions (25 mL) containing 0.02 g of PAID-GSG with different pH values ranging from 3 to 7 were vortexed for 20 min and then centrifugated for 10 min and content of mefenamic acid was measured in supernatant liquid at 280 nm by UV-Vis spectrometry.
In method procedure for best elution solvent, a set of micro-tubes each containing 1 mL of 2 µg/mL solution of mefenamic acid and 0.01 g PAID-GSG was vortexed for 20 min, centrifugated for 10 min and subsequently the supernatant was decanted. Then 1 mL of elution solvent was added to the polymer, sonicated at 55 ºC for 1 h, then vortexed for 15 min and finally centrifugated. The concentration of mefenamic acid in eluent was determined by injection to HPLC column.

Isotherm studies
Isotherm studies were performed by adding 0.01 g of PAID-GSG to a series of microtest tubes filled with 1 mL diluted solutions of mefenamic acid (10-60 μg/mL) at pH 4. The micro-tubes were vortexed for 20 min. The amount of mefenamic acid at equilibrium q e (mg/g) on PAID-GSG was calculated from the following equation: Equation (1) Where C 0 and C e (mg/L) are initial and equilibrium concentration of the mefenamic acid, respectively, V (L) is the volume of the solution and W (g) is the mass of the PAID-GSG.

Optimization of parameters Effect of pH
The experimental results of mefenamic acid sorption as a function of pH are shown in Figure  2. The best extraction was found to be at pH 4.0. The solubility of mefenamic acid with carboxylic acid group is minimum at acidic pH, therefore higher extraction is achieved. pH values more than 7 were not considered because of solubility of silica gel in alkaline pH.

Effect of adsorption time
To investigate the time required for optimum adsorption of mefenamic acid onto polymer, 0.02 g of polymer was shaken with 10 mL of solution containing 10 mg/L mefenamic acid at pH 4 and different length of time (0.5-20 min). After centrifugation, the concentration Scheme 2. Schematic presentation of PAID-GSG synthesis process. AA (0.2%) and TFA (1%) shows the best result and can be used for optimum removal of the mefenamic acid from PAID-GSG.

Adsorption isotherm
The kinetic sorption of mefenamic acid at pH 4 is shown in (Figure 3). As shown 2 min was required to reach 100% sorption saturation. The profile of mefenamic acid uptake on the sorbent reflects the good accessibility of binding sites in the PAID-GSG. Maximum sorption capacity can be defined by Langmuir isotherm which is valid for monolayer sorption on PAID-GSG surface with number of identical active sites. In Langmuir isotherm it is assumed that the of mefenamic acid in supernatant solution was determined by UV-Vis spectrometry. As shown in (Figure 3) about 2 min was required to reach 100% sorption saturation, but to ensure that sorption is complete 20 min was chosen as the optimum adsorption time.

Effect of different eluent solvents on the recovery
The effect of various eluent on the extraction recovery of mefenamic acid was also investigated. The eluents used were methanol, AA and TFA. Presence of AA and TFA in eluent improved the recovery (26-28). Amongst the eluents listed in Table 1, methanol containing Equation (2) Where q max and K L are the maximum mefenamic acid sorption capacity corresponding to complete monolayer coverage on the PAID-GSG surface and the Langmuir constant, respectively.
The linear regression equation in ( Figure  4) suggested that the homogeneous sites for mefenamic acid were formed in the PAID-GSG .From the slope (1/q max ) and intercept (1/q max .K L ) of this line, K L and q max were calculated to be  0.98 L/mg and 7.0 mg/g, respectively.
The essential characteristic of the Langmuir model is expressed with R L : Equation (3) The calculated R L value was 0.017 that is in the range of 0-1 that indicates the adsorption nature is favourable (0<R L <1) (29-30).

Determination of mefenamic acid in capsules
The proposed SPE-HPLC procedure was successfully applied for determination of mefenamic acid in capsules. Capsules of mefenamic acid (20 capsules) bought from local pharmacy (250 mg capsules from Amin pharmaceutical company) were weighed accurately and powdered using a mortar and pestle. A portion of the powder equal to the weight of one capsule was dissolved in methanol and was shaken for 2 hours and then it was sonicated for 15 minutes in order to aid dissolution and then filtered. The filtrate was diluted by water and a 2 µg/mL solution was prepared. The method procedure was performed on this sample for extraction and determination of mefenamic acid content. The concentration of mefenamic acid was obtained from calibration curve. Results are shown in Table 2. The results shown in this table indicate the applicability of this procedure with high recovery.

Determination of mefenamic acid in human plasma and urine
Human blood was collected from thoroughly controlled voluntary blood donors. Each bloodcontaining unit was separately controlled and found negative for Hbs antigen and HIV I, II and hepatitis C antibodies. The red blood cells were separated from the plasma by centrifugation at 4500×g for 30 min at room temperature, then filtered and frozen at -20 ºC. Before use, the plasma was thawed for 1 h at 37 ºC. In order to reduce the matrix effect, the plasma was deproteinized by mixing it with equal volume of acetonitrile (31), vortexed for 3 min, centrifugated for 5 min at 1500×g, filtered (24 mµ) spiked with mefenamic acid. A spiked urine sample was prepared directly from urine sample. Spiked samples of plasma and urine were subjected to the proposed extraction procedure and injected to HPLC column. Results are given in Table 2, which demonstrates the reasonability and reliability of the proposed extraction procedure.
Analytical performance of the method Six replicate determination of a 2 µg/mL solution of mefenamic acid gave a relative standard deviation (RSD %) of 0.65 %. The limit of detection (LOD)corresponding to three times the blank standard deviation divided by slope of calibration curve was found to be 0.46 µg/L. The limit of quantification (LOQ) corresponding to ten times the blank standard deviation was found to be 1.5 µg/L. The concentration of mefenamic acid were obtained from calibration curve with regression equation of A=22.88C+24.278, in which A is the area under the peak and C is the concentration of mefenamic acid (µg/mL) with correlation of coefficient (R 2 =0.9919).

Conclusions
A novel polymer grafted to silica gel (PAID-GSG) was synthesized and used for solid phase extraction of mefenamic acid followed by HPLC determination. The results indicate that the suggested method has some advantages such as little solvent consumption, high chemical stability, simplicity, extraction efficiency, less time consuming compared to other adsorbents. The sorption rate of the investigated mefenamic acid on the PAID-GSG was excellent (about 2 min). The PAID-GSG can be applied to the extraction of trace mefenamic acid in human plasma, urine and pharmaceutical samples with satisfactory results.